Sphingomyelin (SM) is one of the most abundant phospholipids in mammalian plasma. In a recent coronary heart disease (CHD) case- control study, we found that plasma SM levels and SM/(SM+PC) ratios in cases (n=279) were significantly higher than those of controls (n=277). Plasma SM levels and SM/(SM+PC) ratios were discovered to have independent predictive values for CHD after adjusting for age, plasma lipids, smoking, diabetes, hypertension, fibrinogen, and C-reactive protein. Serine palmitoyl-CoA transferase (SPT) catalyzes the initial reaction of sphingolipid synthesis, i.e., the condensation of palmitoyl- CoA with serine to yield the long-chain base (LCB) 3-ketosphinganine. The activity of SPT appears to influence SM levels in several tissues including liver, lung, and aorta, as well as plasma. We found that hepatic SPT activity in apolipoprotein E-deficient mice (apoE KO), a mouse model of atherosclerosis, was increased two fold over that in wild type (Wt) mice, in part explaining the increase of SM levels in very low density lipoprotein (VLDL) and LDL. After mammalian sphingomyelinase (SMase) treatment, the VLDL and LDL isolated from plasma of apoE KO mice had a tendency to aggregate, a process that is thought to stimulate arterial foam cell formation. Recently, two subunits of SPT, LCB1 and LCB2, have been cloned. Heterozygous LCB2 KO mice showed a reduction of plasma SM levels, indicating that LCB2 is directly involved in SM metabolism. Our working hypothesis is that SM- rich lipoproteins are involved in the atherogenic process by deposition and retention in artery walls as a result of local interaction with SMase. It is notable that SM has a unique biosynthetic pathway with completely different regulation from cholesterol biosynthesis. The goal of this project is to investigate the roles of SPT and SM in lipoprotein metabolism and the atherosclerosis. The major experimental emphasis will be on the use of transgenic and gene knock-out mice. The specific aims are: 1) to investigate the roles of SM in the process of atherosclerosis by crossing heterozygous LCB2 KO mice with apoE KO mice, using a specific SPT inhibitor in apoE KO ones; and using an SM- rich diet in LDL receptor KO mice; 2) to establish liver-specific LCB1 and LCB2 transgenic mice for investigating the roles of liver LCB 1 and LCB2 gene overexpression in maintaining plasma SM levels and atherosclerosis; and 3) to generate liver-specific LCB2 KO mice for investigating the role of SPT in SM metabolism and atherosclerosis. This project will provide new information on the regulation of SM metabolism and on the relationship between SM and SPT, as well as between SM and atherosclerosis. If the enrichment of SM in plasma has pro-atherogenic activity, as anticipated, then the inhibition of SPT, the key enzyme for SM biosynthesis, could be an important treatment for atherosclerosis.